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A cone snail venom peptide, μO§-conotoxin GVIIJ from Conus geo-graphus, has a unique posttranslational modification, S-cysteinylated cysteine, which makes possible formation of a covalent tether of peptide to its target Na channels at a distinct ligand-binding site. μO§-conotoxin GVIIJ is a 35-aa peptide, with 7 cysteine residues; six of the cysteines form 3 disulfide cross-links, and one (Cys24) is S-cysteinylated. Due to limited availability of native GVIIJ, we primarily used a synthetic analog whose Cys24 was S-glutathionylated (abbreviated GVIIJSSG). The peptide-channel complex is stabilized by a disulfide tether between Cys24 of the peptide and Cys910 of rat (r) Nav1.2. A mutant channel of rNav1.2 lacking a cysteine near the pore loop of domain II (C910L), was >103-fold less sensitive to GVIIJSSG than was wild-type rNav1.2. In contrast, although rNav1.5 was >104-fold less sensitive to GVIIJSSG than Nav1.2, an rNav1.5 mutant with a cysteine in the homologous location, rNav1.5[L869C], was >103-fold more sensitive than wild-type rNav1-5. The susceptibility of rNav1-2 to GVIIJSSG was significantly altered by treating the channels with thiol-oxidizing or disulfide-reducing agents. Furthermore, coexpression of rNavβ2 or rNavβ4, but not that of rNavβ1 or rNavβ3, protected rNav1.1 to -1.7 (excluding Nav1.5) against block by GVIIJSSG- Thus, GVIIJ-related peptides may serve as probes for both the redox state of extracellular cysteines and for assessing which Navβ- and Navα-subunits are present in native neurons. [ABSTRACT FROM AUTHOR]